PROJECT ABSTRACT HIV-1 associated neuroinflammation and neurotoxicity lead to cognitive impairments (HIV-1-associated neurocognitive disorders or HAND) even in those under suppressive antiretroviral therapy (ART). As people living with HIV-1 age, a compounding effect is occurring, with age associated dementia added to HAND, leading to a complex web of neurocognitive deficit. This will have tremendous implications for health care systems not only in the USA but also in the developing world. How HAND develops, and how it could be modified remain mysterious, largely because It has been very difficult to study HIV-1 infection and HAND in the human brain. Over the past few years new developments in stem cell technologies have permitted the differentiation of ?cerebral organoids? from induced pluripotent stem cells (iPSCs), and these cultures can be grown in vitro in conditions that promote three-dimensional expansion of neuroectoderm, in cerebral organoid or ?miniature brain? forms. Cerebral organoids are heterogeneous and form a variety of brain regions, including ventral forebrain, cerebral cortex, hippocampus, and mid- and hindbrain boundary. They exhibit neurons that are functional and capable of electrical excitation, and develop microglia. These brain organoids also resemble human cortical development at the gene expression level, and allow in depth analysis of neural networks, cell behavior, drug screening, disease modeling, and variations in brain development. While brain-region composition varies in organoids from different iPSC lines, regional gene-expression patterns remain largely reproducible across individuals. These create unparalleled new opportunities to study HIV-1 infection of the brain. We propose to develop our iPSC derived organoid model which incorporates microglia, into one that better represents an adult mature brain that can support robust HIV-1 infection. With this model, we can test whether different viruses lead to differential neurotoxicity and if cells other than microglia can be latently infected with virus. We will test how individual proteins from the virus, including HIV-1 Tat causes neurological damage, and how ART or drugs such as Didehydro-Cortistatin A (dCA), which cross-neutralizes Tat activity, affects the process. Interestingly, exposure to Tat also potentiates cocaine-mediated reward mechanisms, which further promotes HAND, revealing a complex web of interactions between HIV-1 infection and drugs of substance abuse. We will determine how Tat and cocaine collaborate in neurological damage and determine if dCA can reverse it. As cerebral organoids provide a model for HIV-1 latency in the brain we can test whether administration of dCA, can ?block-and-lock? any residual virus, and finally whether HIV-1 specific cytotoxic T cells (CTL) can eliminate virus in the organoid. Together, these studies promise to provide novel insights into the pathogenesis of HAND, Tat mediated neurotoxicity, effects of cocaine, and the potential link between these processes. Finally, the model promises to add exciting findings on HIV-1 latency in the brain, and if it can be silenced or eliminated.